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L V Kozlov

Publications and source records attributed to L V Kozlov.

14 recordsLinked to original sources
PubMed1979

[Principle of free energies linearity in chymotrypsin catalysis].

The applicability of the previously described principle of free energies linearity for semispecific substrates of chymotrypsin for the derivatives of acetylphenylalanine, acetyltryptophane and acetyltyrosine was demonstrated. The pH dependence of the parameters of a linear equation describing the relationship between the free energy of hydrolysis and the logarithmus of the kcat/Km(app) ratio suggests that at optimal pH the stability of the activation barrier of enzyme acetylation for all semispecific substrates is observed.

Chymotrypsin↗
PubMed1979

[Dependence of pepsin conformational states on pH and temperature].

A conformational behaviour of pepsin depending on pH and temperature was studied by circular dichroism, differential UV-spectroscopy, calorimetry and enzymatic hydrolysis kinetics. A subtile conformational transition of the enzyme accompanied by changes in the physico-chemical and enzymatic properties of the protein was observed within the temperature interval of 15--40 degrees and within the pH range of 1,1--5,6. The range of pepsin heat denaturation was studied. A diagram of pepsin conformational states under different values of pH and temperature was built.

Calorimetry↗
PubMed1979

[Nature of the intermediate compound formed during catalysis by pepsin].

The applicability of the free energies linearity principle to catalysis by pepsin may indicate that the mechanism of formation of a covalent pepsin intermediate with the second (amine or alcohol) part of the hydrolyzed substrate is a true one. The choice has been made from consideration of three possible types of mechanisms covalent binding, complex with a nonionized NH2-group and the absence of an activated intermediate.

Enzyme Activation↗
PubMed1979

[Steady-state kinetics of reactions catalyzed by serine sulfhydrases of Saccharomyces serevisiae].

The steady-state kinetics of the two substrate reaction of L-cysteine desulfation in the presence of 2-mercaptoethanol catalyzed by serine sulfhydrase from bakers yeast -- a pyridoxal phosphate-containing enzyme of the beta -- substituting lyase type -- were studied. Highly purified enzyme preparations (approximately 90% purity) of Saccharomyces cerevisiae with specific activity of 25 mumoles of H2S per 1 hr per mg of protein were used. The values of V, KS1, KS2 and alpha were calculated from the initial rates of the reaction under constant concentration of L-cysteine (S1) and variable concentration of 2-mercaptoethanol (S2) and vice versa. The data obtained suggest that under conditions of a two-substrate reaction catalyzed by serine sulfhydrase and in case of beta-cyanoalanine synthase of blue lupin the substrate binding to the enzyme is interdependent and obeys a unordered mechanism with o formation of a ternary aminosubstrate-pyridoxal phosphateenzyme-cosubstrate complex (alpha = 2.6).

Cystathionine beta-Synthase↗
PubMed1978

[Effect of soluble matrix on the stability of modified alpha-chymotrypsin].

alpha-Chymotrypsin preparations covalently bound by Shiff bases with water soluble oxidated dextran and alginate are obtained to study the effect of charged and neutral matrices on the enzymes stability under their modification by polymers. Water soluble enzyme preparations show a catalytic activity and have a slightly enhanced thermostability. Thermostability of alpha-chymotrypsin modified by a negatively charged polymer is increased owing to the reducing of activation entropy of the denaturation reaction, while the increase of the stability of neutral polymer (dextran) modified enzyme is due to the increase of activation enthalpy of the denaturation reaction.

Alginates↗
PubMed1977

[Enzymatic properties of immobilized beta-galactosidase from Curvularia inaequalis].

beta-Galactosidase (EC 3.2.1.23) from fungus Curvularia inaequalis was modified by active brilliant orange KH and adsorbed on DEAE-Sephadex A-50. The lactose hydrolysis was studied in a continous flow on the column packed with the immobilized enzyme. The pH and temperatures optima for the substrate hydrolysis by the immobilized enzyme were shown to remain unchanged. A certain destabilizing effect of the matrix on the enzyme resistance to hear denaturation was observed. The activation parameters of denaturation of the native enzyme as well as those of the dye-modified and immobilized preparations were determined.

Chemical Phenomena↗
PubMed1977

[On the changes in alpha-chymotrypsin stability after its modification by polyelectrolytes].

Reversible thermal denaturation of alpha-chymotrypsin, its electrostatic complexes with carboxyl-containing polymers and the enzyme covalently bound with those polymers were studied. It was shown that the enzyme stability is affected by matrix, which manifests itself in a simultaneous decrease in enthalpy and enthropy of the reversible denaturation process and a simultaneous decrease in activation enthalpy and enthropy of denaturation. Modification and complexing of chymotrypsin with polymers has practically no effect on the activation parameters of renaturation. Differences in the original states of alpha-chymotrypsin and its derivatives and similarity of their activated states are proposed. The formation of ionic complexes of enzyme or covalent binding to polymers results in alteration of the protein native state similar to the denaturated state.

Chymotrypsin↗
PubMed1976

[Modification and immobilization of beta-galactosidase].

beta-galactosidase from fungus Curvularia inaequalis was modified by a chlortriazin dye active bright-orange KH. The modified enzyme contained two molecules of dye per one molecule of protein. The incorporation of six sulfuric groups with remains of the dye resulted in a slight decrease of the acid protein isoelectric point. The catalytic activity of the modified protein remains practically unchanged. The coloured protein is firmly absorbed on anionites. Preparations of immobilized beta-galactosidase were obtained by adsorption on anionites.

Catalysis↗
PubMedDate not supplied

[pH-dependence of the mechanism of pepsin action].

N-Acetyl-L-phenylalanine inhibition of the peptic hydrolysis of N-acetyl-L-phenylalanine-L-tyrosine over the pH range 2-4.5 was studied. The mixed character of inhibition which was partially competitive and partially non-competitive allowed us to infer that the separate steps of the enzymatic hydrolysis were pH dependent. The orderliness of the dissociation of the triple enzyme-product-product complex was also pH dependent. The group with pKa approximately 3 influenced the mechanisms of pepsin hydrolysis as strongly as in the case of pepsin catalyzed oxygen isotopic exchange in the acyl amino acid carboxyl group.

Binding Sites↗
PubMedDate not supplied

[Influence of pH and thermal treatment on the intramolecular mobility of pepsin].

In connection with a hypothesis of segmental flexibility of pepsin and its derivative aminopepsin (containing 3-aminotyrosine residues), a dynamic behaviour of pepsin and dansylated aminopepsin in aqueous solutions at pH 1.0--8.3 in investigated. It is shown that a dynamic structure of pepsin at pH 2.5--6.0 is almost invariant and very close to a structure studied in detail at pH 5.5. The lowering of a solution pH value from 2.5 to 1.0 is accompanied by labilization of pepsin and dansylated aminopepsin structures. Denaturation phenomena after thermal treatment of dansylated aminopepsin are also studied. An incubation of the latter in 0.1 M acetate buffer at pH 5.5 and temperature 50--80 degrees C with a subsequent cooling leads to irreversible conformational changes. The character of these changes is essentially dependent upon the incubation conditions.

Hydrogen-Ion Concentration↗
PubMedDate not supplied

[The fluorescence of pepsin conjugates with DNS-chloride].

Parameters of rotational relaxation of pepsin conjugated in neutral and slightly alkaline solutions with a fluorescent label 1-dimethylaminonaphthalene-5-sulphonyl chloride (DNS-Cl) are measured by a fluorescence polarization method. It is shown that the globule of pepsin denatured and loose at lakaline pH values converts into a compact form after transfer to acidic solution. The compactness of this new form is close to that of native inhibited pepsin. A new globule is distinguished from the native by the absence of segmental flexibility. Conjugated with a DNS at pH less than or equal to 7.0 pepsin relaxes in solution as catalytically active dansylated aminopepsin (DNS-3-aminotyrosine pepsin). Evidence is presented that these conjugates are also characterized by segmental flexibility.

Animals↗
PubMedDate not supplied

[Intramolecular mobility of pepsin].

By modifying four tyrosine residues in pepsin a derivative (aminopepsin) is obtained which is capable to conjugate with a fluorescent label DNS-Cl without loss of the catalytic activity. Rotational relaxation times of native pepsin and dansylated aminopepsin (DAP) are measured by a fluoresence polarization method. The values obtained are shown to be lower than those calculated for arigid pepsin globule. Possible sources of the obtained difference are discussed. A reversible or covalent blocking of pepsin or DAP active centres by specific inhibitors leads to an increase in rotational relaxation time values for these proteins reaching the magnitude which is very close to that calculated for a model of rigid pepsin. Brownian relaxation of pepsin and DAP reduced by beta-mercaptoethanol and of pepsinogen and some fragments of pepsine macromolecule in aqueous solutions is invigated as well. The results are intepreted as representing an intramolecular mobility or segmental flexibility of pepsin and DAP. With the use of the obtained and X-ray data a segmental model of dynamic pepsin structure is suggested. On the basis of this model some conclusions are drawn concerning a localization of a polypeptide which is split off from the N-terminus of pepsinogen during its activation. A possible role of segmental flexibility in the catalytic action of pepsin is considered.

Animals↗